Enhancing anti-tumor response in melanoma cells with defective sting signaling

ABSTRACT

Disclosed herein is a method for enhancing antitumor T cell responses in subjects. The method involves administering to the subject in need thereof a composition comprising a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. This method is particularly useful in subjects with deficient STING expression in the tumor cells. Therefore, also disclosed is a method for treating a tumor in a subject that involves detecting in a biopsy sample from the subject reduced STING expression, reduced cGAS expression, or a combination thereof; and then administering to the subject a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. The method can further involve administering to the subject a therapeutically effective amount of a STING agonist. The method can further involve administering to the subject tumor infiltrating lymphocytes (TILs), such as HLA-matched TILs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending InternationalApplication No. PCT/US2019/042788, filed Jul. 22, 2019, which claimsbenefit of U.S. Provisional Application No. 62/702,195, filed Jul. 23,2018, and Application Ser. No. 62/712,561, filed Jul. 31, 2018, whichare hereby incorporated herein by reference in their entireties.

BACKGROUND

Stimulator of interferon genes (STING) is an endoplasmicreticulum-resident signaling molecule. It is responsible for controllingthe transcription of several host defense genes, including type I IFNsand pro-inflammatory cytokines, in response to recognition of cytosolicDNA species or cyclic dinucleotides. Recent studies have indicated thatSTING signaling is the major innate immune pathway involved in thegeneration of a spontaneous antitumor T cell response. Mice lackingSTING cannot generate efficient antitumor T cell responses and rejectmelanoma tumor growth. Based on this finding, many STING agonists havebeen developed to utilize STING activation as a cancer therapy. Suchagonists have been found to be experimentally useful in inducing robusttumor control through the host immune cell activation. While STINGactivation has been extensively investigated in antigen presenting cells(in particular dendritic cells), little is known regarding itsactivation in tumor cells.

SUMMARY

Disclosed herein is a method for enhancing antitumor T cell responses insubjects, such as those receiving adoptive cell transfer (ACT) of tumorinfiltrating lymphocytes (TIL). The method involves administering to thesubject in need thereof a composition comprising a demethylating agentin an amount effective to demethylate STING proteins in the tumor cells,such as melanoma cells. This method is particularly useful in subjectswith deficient STING expression in the tumor cells. Therefore, alsodisclosed is a method for treating a tumor in a subject that involvesdetecting in a biopsy sample from the subject reduced STING expression,reduced cGAS expression, or a combination thereof; and thenadministering to the subject a demethylating agent in an amounteffective to demethylate STING proteins in the tumor cells. The methodcan further involve administering to the subject a therapeuticallyeffective amount of a STING agonist. The method can further involveadministering to the subject tumor infiltrating lymphocytes (TILs), suchas HLA-matched TILs.

Also disclosed is a method for enhancing TIL function that involvesculturing a tumor sample from a subject in the presence of i) ademethylating agent in an amount effective to demethylate STING proteinsin the tumor cells and ii) a STING agonist in an amount effective toincrease the antigenicity of the tumor cells and/or upregulatingexpression of MHC molecules, and then exposing TILs to these modifiedtumor cells to enhance their function (i.e. killing and cytokineproduction) and further expansion for subsequent adoptive transfer tothe cancer patient. In preferred embodiments, the TILs are HLA matchedto the tumor sample from the subject.

Also disclosed is a method of treating a subject that involves producingTILs according to the disclosed methods and then adoptively transferringthe TILs to the subject. Also disclosed is a method of treating a cancerin a subject that involves detecting in a tumor sample from the subjectreduced STING expression, reduced cGAS expression, or a combinationthereof, and then administering to the subject a therapeuticallyeffective amount of TILs produced according to the disclosed methods.For example, in some embodiments of the disclosed methods, the subjecthas a cancer, and the method treats the cancer.

Downstream induction of CXCR3-binding chemokines such as CXCL10 andCXCL9 in melanoma cell lines following their stimulation with the STINGagonist has at least three important implications. First, it could beused to recruit higher numbers of T cells into the tumors that lack Tcell infiltration and therefore increase the likelihood of patientsresponding to current immune checkpoint antibody therapies. Second, asame strategy could be used in TIL-based therapies prior to tumorresection and TIL expansion to attract higher numbers of tumor-specificT cells into the tumors with the aim of increasing the probability ofsuccessful expansion of tumor-reactive TIL ex vivo. A third implicationwould also be in adoptive T cell therapy where STING agonist-mediatedCXCL10 induction in tumor cells could be used to improve TIL traffickinginto the tumor sites.

In some embodiments, the tumor of the disclosed compositions and methodsis a solid tumor. In some cases, the tumor is a melanoma, ovariancancer, breast cancer, or colorectal cancer. The cancer can bemetastatic, recurrent, or a combination thereof. In some embodiments,these TILs are administered back to the subject. In some of theseembodiments, the subject is treated with a demethylating agent and aSTING agonist.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are example blots showing STING and cGAS expression inmelanoma cell lines.

FIG. 2 illustrates the activation of STING pathway in NK92 cells(positive control) with 2′3′-cGAMP.

FIG. 3 is a bar graph showing induction of CXCL-10 expression in NK92cells after 4 h treatment with 2′3-cGAMP.

FIG. 4A is an example blot showing pIRF3, IRF3, and β-actin expressionin NK92 and human melanoma cell lines 164, WM9, WM39, A375, 1366, and2032 with and without treatment with 2′3′-cGAMP. FIGS. 4B and 4C are bargraphs showing CXCL-10 (FIG. 4B) and IFN-β expression in NK92 and humanmelanoma cell lines 164, WM9, WM39, A375, 1366, and 2032 with andwithout treatment with 2′3′-cGAMP.

FIGS. 5A and 5B are bar graphs showing expression of CXCL-10 (FIG. 5A)and IFN-β (FIG. 5B) in WM9 and WM39 cell lines after 4 h & 24 htreatment with 2′3′-cGAMP.

FIG. 6 is a bar graph showing expression of CXCL-10 in STING positivemelanoma cell lines after 4 h & 24 h treatment with 2′3-cGAMP.

FIGS. 7A to 7E show DNA demethylation partially recapitulated STING andcGAS expression in human melanoma cell lines. FIG. 7A is a bar graphshowing cGAS relative expression level in SK-MEL-23, A375, and SK-MEL-5cells with and without 5AZADC treatment. FIG. 7B is an example blotshowing STING, cGAS and β-actin expression in A375, G361, MeWO, andSK-MEL-5 cells with and without 5AZADC treatment. FIG. 7C are exampleimages of STING and cGAS in A375, G361, MeWO, and SK-MEL-5 cells withand without 5AZADC treatment. FIGS. 7D and 7E are bar graphs showingIFN-β (FIG. 7D) and CXCL10 (FIG. 7E) fold changes in SK-MEL-24, A375,G361, MeWo, and SK-MEL-5 cells with and without 5AZADC treatment, withLipo or dsDNA90.

FIGS. 8A to 8D show reconstitution of STING expression in 5AZADC-treatedmelanoma cells and STING-dependent CXCL-10 induction following2′3′-cGAMP stimulation. FIG. 8A is an example blot of STING and β-actinexpression in 2032, 266-4, and 1361 A cells with and without 5AZADCtreatment. FIGS. 8B to 8D are bar graphs showing CXCL-10 expression in2032 (FIG. 8B), 266-4 (FIG. 8C), and 1361 A (FIG. 8D) cells with andwithout 5AZADC treatment.

FIGS. 9A to 9F are bar graphs showing CXCL-10 induction in 1366 (FIG.9A), 526 (FIG. 9B), 164 (FIG. 9C), A375 (FIG. 9D), WM3629 (FIG. 9E), andWM9 (FIG. 9F) melanoma cells with and without 5AZADC treatment.

FIG. 10 is a bar graph showing activation of STING signaling in MART-1pulsed WM39 melanoma cells results in increased IFN-γ secretion whencultured with V40195 TIL.

FIGS. 11A to 11I are bar graph showing IFN-γ (FIGS. 11A-11C), CXCL-10(FIGS. 11D-11F), and IFN-β (FIGS. 11G-11I) expression in co-cultures ofMART-1 pulsed WM39 with HLA-matched TILs.

FIGS. 12A to 12I are bar graph showing IFN-γ (FIGS. 12A-12C), CXCL-10(FIGS. 12D-12F), and IFN-β (FIGS. 12G-12I) expression in co-cultures ofWM39, MART-1 pulsed WM39 or WM3629 with V40195 TIL.

FIGS. 13A and 13B show activation of STING pathway in human melanomasimproves their cytotoxic T lymphocyte-mediated lysis.

FIG. 14 shows MHC-I/HLA-A.B.C expression in melanoma cell lines aftertreatment with 2′3′ cGAMP. STING activation in human melanoma cell linesinduces upregulation of MHC 1 (HLA-A.B.C).

FIGS. 15A to 15G show identification of melanoma cell lines with intactSTING signaling. FIG. 15A shows immunoblot analysis of STING and cGASexpression in a series of human melanoma cell lines. NK-92 was used as apositive control for the expression of STING and cGAS. Twenty μg ofwhole-cell lysate was used and β-actin was analyzed as a loadingcontrol. FIGS. 15B and 15C show ratio of total STING relative to β-actin(FIG. 15B), and ratio of total cGAS relative to β-actin (FIG. 15C) foreach cell line were quantified using ImageJ software. FIG. 15D showsimmunoblot analysis of p-IRF3 and total IRF3 in five STING-positive(WM164, WM9, WM39, A375 and WM1366) and one STING-negative (WM2032)human melanoma cell lines after 4 h stimulation with 2′3′-cGAMP orlipofectamine. Twenty μg of whole-cell lysate was used and β-actin wasanalyzed as loading control. FIG. 15E shows ratio of p-IRF3 relative toIRF-3 for 2′3′-cGAMP stimulated cell lines were quantified using ImageJsoftware. FIGS. 15F and 15G show induction of CXCL10 (FIG. 15F) andIFN-β (FIG. 15G) in cell culture supernatants of indicated humanmelanoma cells after.

FIGS. 16A to 16E show STING signaling is required in melanoma cells foragonist-induced improved antigenicity. FIG. 16A shows immunoblotanalysis of p-IRF3 and total IRF3 in 526-MEL and WM39 melanoma cellsafter stimulation with 2′3′-cGAMP. FIGS. 16B and 16C show induction ofCXCL10 (FIG. 16B) and IFN-β (FIG. 15C) in 526-MEL and WM39 cells after24 h stimulation with 2′3′-cGAMP measured using ELISA. FIGS. 16D and 16Eshow 526-MEL and WM39 cells were co-cultured with TIL 19 (FIG. 16D) andTIL 195 (FIG. 16E) for 24 h with or without 2′3′-cGAMP. IFN-γ levels insupernatants were measured using ELISA.

FIGS. 17A to 17F show activation of STING pathway in human melanoma celllines improves cytotoxic T lymphocyte-mediated lysis. FIGS. 17A to 17Eshow ⁵¹Cr cytotoxicity assays using WM39 (FIG. 17A), MART-1 pulsed WM39(FIG. 17B), WM39+w6/32 (FIG. 17C), WM3629 (FIG. 17D), and 526-MEL (FIG.17E) cells as target cells and TIL 195 as effector cells at theindicated effector/target (E/T) ratios with or without 2′3′-cGAMP. Datarepresent the mean±SEM of quadruplicate wells. FIG. 17F show lyticactivity of TIL 195 against different agonist-treated and untreatedmelanoma targets was measured in lytic units (10⁶ divided by the numberof effector cells required to cause 20% lysis of 5×10³ tumor cells).

FIGS. 18A and 18B show STING activation in human melanoma cell linesinduces up-regulation of MHC class I (HLA-A.B.C). FIG. 18A showsrepresentative histograms of HLA-A.B.C expression on fourSTING-defective (1205Lu, WM266-4, WM2032 and 526-MEL) and fourSTING-intact (WM9, WM3629, A375 and WM39) human melanoma cell lines withor without 2′3′-cGAMP stimulation. FIG. 18B shows mean fluorescenceintensity (MFI) of HLA-A.B.C on indicated human melanoma cells.

FIGS. 19A to 19F shows knockdown of STING blocks agonist-inducedupregulation of MHC class I in melanoma cells. WM39 cells were stablytransduced with a lentiviral shRNA specific for STING (sh-STING) ornon-target shRNA (sh-control). FIG. 19A shows immunoblot analysis ofSTING expression in WM39, sh-control, and sh-STING cells. Twenty μg ofwhole-cell lysate was used and β-actin was analyzed as a loadingcontrol. FIG. 19B shows immunoblot analysis of p-IRF3 and total IRF3 inWM39, sh-control and sh-STING cells after stimulation with 2′3′-cGAMP orlipofectamine. FIGS. 19C and 19D show induction of CXCL10 (FIG. 19C) andIFN-β (FIG. 19D) in WM39, sh-control and sh-STING cells afterstimulation with 2′3′-cGAMP or lipofectamine. FIG. 19E showsrepresentative histograms of HLA-A.B.C expression on indicated cellswith or without 2′3′-cGAMP stimulation. FIG. 19F shows mean fluorescenceintensity (MFI) of HLA-A.B.C on indicated cells. Data are represented asmean±SEM. **p<0.01; ****p<0.0001 (Student's t test).

FIGS. 20A to 20C show STING is essential for agonist-induced enhancedantigenicity in melanoma cells. FIG. 20A shows WM39, sh-control andsh-STING cells were co-cultured with TIL 195 for 24 h in the presence orabsence of 2′3-cGAMP. IFN-γ levels in supernatants were measured usingELISA. FIG. 20B shows ⁵¹Cr cytotoxicity assay using WM39, sh-control andsh-STING cells as target cells and TIL 195 as effector cells at theindicated effector/target (E/T) ratios with or without 2′3′-cGAMP. Datarepresent the mean±SEM of quadruplicate wells. FIG. 20C shows lyticactivity of TIL 195 against indicated targets with or without 2′3′-cGAMPstimulation was measured in lytic units.

FIG. 21 shows blockade of IFNAR inhibits agonist-induced upregulation ofMHC class I.

FIGS. 22A and 22B shows in vitro stimulation of SEAP (FIG. 22A) andIFN-β (FIG. 22B) with 2′3′-cGAMP.

FIG. 23 shows in vitro stimulation of IFN-β with 2′3′-cGAMP.

FIG. 24 shows Activation of STING in B16 melanoma cell lines inducesup-regulation of MHC class I.

FIGS. 25A to 25E show loss of STING in B16-ISG accelerates tumor growthat early time points.

FIGS. 26A and 26B show loss of STING in B16-ISG cells does not affecttheir proliferation in vitro.

FIGS. 27A to 27E show loss of STING in B16-ISG cells alters numbers andphenotype of TIL in vivo.

FIGS. 28A and 28B show loss of STING in B16-ISG cells alters numbers andphenotype of TIL in vivo.

FIGS. 29A and 29B show intratumoral 2′3′-cGAMP delays tumor growth ofB16 tumors in wild-type C57BL/6 mice.

FIG. 30 shows intratumoral 2′3′-cGAMP delays tumor growth of B16 tumorsin wild-type C57BL/6 mice.

FIGS. 31A to 31D show reconstitution of STING expression through DNAdemethylation can rescue agonist-induced STING signaling in melanomacell lines.

FIG. 32 shows IFN-β induction in 5AZADC treated melanoma cell lines inresponse to stimulation with 2′3′-cGAMP.

FIG. 33 shows IFN-β levels in 5AZADC treated 1205-Lu cell line inresponse to stimulation with 2′3′-cGAMP.

FIG. 34A to 34D show MHC class I surface expression in 5AZADC treatedmelanoma cell lines in response to stimulation with 2′3′-cGAMP.

FIG. 35A shows cGAS has 8 probes on the EPIC chip. The 6 first probesall show a similar methylation pattern across the 16 melanoma celllines. The bar plot on top shows that the first 6 probes are negativelycorrelated to the protein level of cGAS. 526-MEL, A375 and WM39 show ahigh degree of methylation. WM35, SBCL2, SK-MREL-28, WM858 and WM3629show some degree of methylation while the rest are show littlemethylation. FIG. 35B shows the correlation between the averagemethylation for the 6 first probes and the protein expression of cGAS.It is clear that in most of the cell lines, the expression of cGAS isepigenetically regulated by methylation. A clear exception is 888-MELthat shows low methylation and also low protein expression of cGAS.

FIGS. 36A and 36B shows STING is represented by 18 probes on the EPICchip. Probes 5-15 show a negative correlation with STING proteinexpression. In probes 5-10, WM266-4, WM239A, WM2032, 888-MEL and SBCL2show high degree of methylation.

FIG. 36B shows that these four cell lines have no expression of STINGprotein. Only cell lines with no methylation express STING but there arealso cell lines with no methylation and no protein expression.

FIGS. 37A to 37C show a detailed study of probes 13-15 demonstratingthat the expression of STING can be regulated by several CpG sites.Treating 526-MEL and WM858 would not respond to 5AZAtreatment.

FIG. 38 shows suppression of STING and cGAS expression in human melanomacell lines is associated with high levels of DNA methylation in STINGand cGAS gene promoter regions.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, biology, and the like, which arewithin the skill of the art.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the probes disclosed and claimed herein.Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C., and pressure is at or near atmospheric. Standardtemperature and pressure are defined as 20° C. and 1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “sample from a subject” refers to a tissue (e.g., tissuebiopsy), organ, cell (including a cell maintained in culture), celllysate (or lysate fraction), biomolecule derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), or body fluid from asubject. Non-limiting examples of body fluids include blood, urine,plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitialfluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid,saliva, anal and vaginal secretions, perspiration, semen, transudate,exudate, and synovial fluid.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “tumor infiltrating lymphocyte” or “TIL” refers to white bloodcells that have left the bloodstream and migrated into a tumor.

Method

Disclosed herein is a method for enhancing antitumor T cell responses insubjects, such as those receiving adoptive cell transfer (ACT) of tumorinfiltrating lymphocytes (TIL).

Patient Screening

In some embodiments, the disclosed methods involve assaying a biopsysample from the subject for STING expression, cGAS expression, or acombination thereof. In some embodiments, the disclosed methods involveassaying a biopsy sample from the subject for DNA methylation within theregulatory regions of STING and/or cGAS genes.

In some aspects, the method is an immunoassay. Many types and formats ofimmunoassays are known and all are suitable for detecting the disclosedbiomarkers. Examples of immunoassays are enzyme linked immunosorbentassays (ELISAs), radioimmunoassays (RIA), radioimmune precipitationassays (RIPA), immunobead capture assays, Western blotting, dotblotting, gel-shift assays, Flow cytometry, protein arrays, multiplexedbead arrays, magnetic capture, in vivo imaging, fluorescence resonanceenergy transfer (FRET), and fluorescence recovery/localization afterphotobleaching (FRAP/FLAP).

Demethylating Agents

In some embodiments, the disclosed methods involve treating the subjectwith a demethylating agent. Cytidine analogs with demethylating activityare known. Examples of cytidine analogs with demethylating activityinclude, but are not limited to, 5-Azacytidine,5,6-dihydro-5-azacytidine, 1-β-D-arabinofuranosyl-5-azacytidine,5-Aza-2′-deoxycytidine (decitabine) or1-(beta-D-ribofuranosyl)-1,2dihydropropyrimindin-2-one (zebularine).Additional examples of demethylating agents include 5-aza-cytidine(5-aza-C), 5-aza-2′-deoxycytidine (5-aza-dC or decitabine),5-fluoro-2′-deoxycytidine (5-F-dC), Pseudoisocytidine, 2-Hpyrimidinone-1-b-D (2′-deoxyriboside) (Zebularine), Guadecitabine(SGI-110), and Disulfiram.

STING Agonists

In some aspects, the disclosed methods involve treating the subject witha cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING)pathway agonist. In some aspects, the cGAS/STING pathway agonist is2′3′-cyclic-GMP-AMP (2′3′-cGAMP). Additional STING agonists are known inthe art, including those disclosed in U.S. Patent Publication No.2016/0287623 and U.S. Patent Publication No. 2017/0044206, which areincorporated by reference herein for these agonists. In some aspects,the STING agonist is a natural cyclic dinucleotide (CDN), such as2′3′-cGAMP, 3′3′-cGAMP, c-di-AMP, or c-di-GMP. In some aspects, theSTING agonist is a cAIM-derived CDN, such as cAIMP, cAIMP Difluor, orcAIM(PS)2 Difluor (Rp/Sp). In some aspects, the STING agonist is acGAMP-derived CDN, such as 2′2′-cGAMP, 2′3′-cGAM(PS)2 (Rp/Sp), or3′3′-cGAMP Fluorinated. In some aspects, the STING agonist is ac-di-AMP-derived CDN, such as c-di-AMP Fluorinated, 2′3′-c-di-AMP, or2′3′-c-di-AM(PS)2 (Rp,Rp). In some aspects, the STING agonist is ac-di-GMP-derived CDN, such as c-di-GMP Fluorinated or 2′3′-c-di-GMP. Insome aspects, the STING agonist is a c-di-IMP-derived CDN, such asc-di-IMP. In some aspects, the STING agonist is a xanthenone analog,such as DMXAA. In some aspects, the STING agonist is ADU-S100 (MIW815,Aduro Biotech). In some aspects, the STING agonist is MK-1454 (Merck).

Adoptive Cell Transfer

In some aspects, the disclosed methods involve treating the subject withAdoptive Cell Transfer (ACT) of lymphocytes, such as tumor-infiltratinglymphocytes (TILs), such as HLA-matched TILs.

Tumor-infiltrating lymphocyte (TIL) production is a 2-step process: 1)the pre-REP (Rapid Expansion) stage where you the grow the cells instandard lab media such as RPMI and treat the TILs w/reagents such asirradiated feeder cells, and anti-CD3 antibodies to achieve the desiredeffect; and 2) the REP stage where you expand the TILs in a large enoughculture amount for treating the patients. The REP stage requires cGMPgrade reagents and 30-40 L of culture medium. However, the pre-REP stagecan utilize lab grade reagents (under the assumption that the lab gradereagents get diluted out during the REP stage), making it easier toincorporate alternative strategies for improving TIL production.Therefore, in some embodiments, the disclosed TLR agonist and/or peptideor peptidomimetics can be included in the culture medium during thepre-REP stage.

Adoptive cell transfer (ACT) is a very effective form of immunotherapyand involves the transfer of immune cells with antitumor activity intocancer patients. ACT is a treatment approach that involves theidentification, in vitro, of lymphocytes with antitumor activity, the invitro expansion of these cells to large numbers and their infusion intothe cancer-bearing host. Lymphocytes used for adoptive transfer can bederived from the stroma of resected tumors (tumor infiltratinglymphocytes or TILS). They can also be derived or from blood if they aregenetically engineered to express antitumor T cell receptors (TCRs) orchimeric antigen receptors (CARs), enriched with mixed lymphocyte tumorcell cultures (MLTCs), or cloned using autologous antigen presentingcells and tumor derived peptides. ACT in which the lymphocytes originatefrom the cancer-bearing host to be infused is termed autologous ACT. US2011/0052530 relates to a method for performing adoptive cell therapy topromote cancer regression, primarily for treatment of patients sufferingfrom metastatic melanoma, which is incorporated by reference in itsentirety for these methods.

ACT may be performed by (i) obtaining autologous lymphocytes from amammal, (ii) culturing the autologous lymphocytes to produce expandedlymphocytes, and (ii) administering the expanded lymphocytes to themammal. Preferably, the lymphocytes are tumor-derived, i.e. they areTILs, and are isolated from the mammal to be treated, i.e. autologoustransfer.

Autologous ACT as described herein may also be performed by (i)culturing autologous lymphocytes to produce expanded lymphocytes; (ii)administering nonmyeloablative lymphodepleting chemotherapy to themammal; and (iii) after administering nonmyeloablative lymphodepletingchemotherapy, administering the expanded lymphocytes to the mammal.

Autologous TILs may be obtained from the stroma of resected tumors.Tumor samples are obtained from patients and a single cell suspension isobtained. The single cell suspension can be obtained in any suitablemanner, e.g., mechanically (disaggregating the tumor using, e.g., agentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) orenzymatically (e.g., collagenase or DNase).

Expansion of lymphocytes, including tumor-infiltrating lymphocytes, suchas T cells can be accomplished by any of a number of methods as areknown in the art. For example, T cells can be rapidly expanded usingnon-specific T-cell receptor stimulation in the presence of feederlymphocytes and interleukin-2 (IL-2), IL-7, IL-15, IL-21, orcombinations thereof. The non-specific T-cell receptor stimulus can e.g.include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(available from Ortho-McNeil®, Raritan, N.J. or Miltenyi Biotec,Bergisch Gladbach, Germany). Alternatively, T cells can be rapidlyexpanded by stimulation of peripheral blood mononuclear cells (PBMC) invitro with one or more antigens (including antigenic portions thereof,such as epitope(s), or a cell of the cancer, which can be optionallyexpressed from a vector, such as an human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., approximately 0.3 μM MART-1: 26-35 (27 L) orgp100:209-217 (210M)), in the presence of a T-cell growth factor, suchas around 200-400111/ml, such as 300 IU/ml IL-2 or IL-15, with IL-2being preferred. The in vitro-induced T-cells are rapidly expanded byre-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the T-cellscan be re-stimulated with irradiated, autologous lymphocytes or withirradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

In some embodiments, nonmyeloablative lymphodepleting chemotherapy isadministered to the mammal prior to administering to the mammal theexpanded tumor-infiltrating lymphocytes. The purpose of lymphodepletionis to make room for the infused lymphocytes, in particular byeliminating regulatory T cells and other non-specific T cells whichcompete for homeostatic cytokines Nonmyeloablative lymphodepletingchemotherapy can be any suitable such therapy, which can be administeredby any suitable route known to a person of skill. The nonmyeloablativelymphodepleting chemotherapy can comprise, for example, theadministration of cyclophosphamide and fludarabine, particularly if thecancer is melanoma, which can be metastatic. A preferred route ofadministering cyclophosphamide and fludarabine is intravenously.Likewise, any suitable dose of cyclophosphamide and fludarabine can beadministered. Preferably, around 40-80 mg/kg, such as around 60 mg/kg ofcyclophosphamide is administered for approximately two days after whicharound 15-35 mg/m2, such as around 25 mg/m2 fludarabine is administeredfor around five days, particularly if the cancer is melanoma.

Specific tumor reactivity of the expanded TILs can be tested by anymethod known in the art, e.g., by measuring cytokine release (e.g.,interferon-gamma) following co-culture with tumor cells. In oneembodiment, the autologous ACT method comprises enriching cultured TILsfor CD8+ T cells prior to rapid expansion of the cells. Followingculture of the TILs in IL-2, the T cells are depleted of CD4+ cells andenriched for CD8+ cells using, for example, a CD8 microbead separation(e.g., using a CliniMACS<plus>CD8 microbead system (Miltenyi Biotec)).In an embodiment of the method, a T-cell growth factor that promotes thegrowth and activation of the autologous T cells is administered to themammal either concomitantly with the autologous T cells or subsequentlyto the autologous T cells. The T-cell growth factor can be any suitablegrowth factor that promotes the growth and activation of the autologousT-cells. Examples of suitable T-cell growth factors include interleukin(IL)-2, IL-7, IL-15, IL-12 and IL-21, which can be used alone or invarious combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 andIL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12and IL2. IL-12 is a preferred T-cell growth factor.

Preferably, expanded lymphocytes produced by these methods areadministered as an intra-arterial or intravenous infusion, whichpreferably lasts about 30 to about 60 minutes. Other examples of routesof administration include intraperitoneal, intrathecal andintralymphatic. Likewise, any suitable dose of lymphocytes can beadministered. In one embodiment, about 1×1010 lymphocytes to about15×1010 lymphocytes are administered.

The cancer treated by the disclosed compositions and methods can be anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynxcancer, kidney cancer, larynx cancer, liver cancer, lung cancer,malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer,non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesenterycancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer,skin cancer, soft tissue cancer, testicular cancer, thyroid cancer,ureter cancer, urinary bladder cancer, and digestive tract cancer suchas, e.g., esophageal cancer, gastric cancer, pancreatic cancer, stomachcancer, small intestine cancer, gastrointestinal carcinoid tumor, cancerof the oral cavity, colorectal cancer, and hepatobiliary cancer.

The cancer can be a recurrent cancer. Preferably, the cancer is a solidcancer. Preferably, the cancer is melanoma, ovarian, breast andcolorectal cancer, even more preferred is melanoma, in particularmetastatic melanoma.

Combination Therapy

The disclosed compositions and methods can be used in combination withany compound, moiety or group which has a cytotoxic or cytostaticeffect. Drug moieties include chemotherapeutic agents, which mayfunction as microtubulin inhibitors, mitosis inhibitors, topoisomeraseinhibitors, or DNA intercalators, and particularly those which are usedfor cancer therapy. The disclosed compositions and methods can be usedin combination with immunotherapy. For example, in some embodiment, thedisclosed compositions and methods are used in combination with CAR-Ttherapy.

The disclosed compositions and methods can be used in combination with acheckpoint inhibitor. The two known inhibitory checkpoint pathwaysinvolve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4)and programmed-death 1 (PD-1) receptors. These proteins are members ofthe CD28-B7 family of cosignaling molecules that play important rolesthroughout all stages of T cell function. The PD-1 receptor (also knownas CD279) is expressed on the surface of activated T cells. Its ligands,PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on thesurface of APCs such as dendritic cells or macrophages. PD-L1 is thepredominant ligand, while PD-L2 has a much more restricted expressionpattern. When the ligands bind to PD-1, an inhibitory signal istransmitted into the T cell, which reduces cytokine production andsuppresses T-cell proliferation. Checkpoint inhibitors include, but arenot limited to antibodies that block PD-1 (Nivolumab (BMS-936558 orMDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A,MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX-010),Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3(BMS-986016).

Human monoclonal antibodies to programmed death 1 (PD-1) and methods fortreating cancer using anti-PD-1 antibodies alone or in combination withother immunotherapeutics are described in U.S. Pat. No. 8,008,449, whichis incorporated by reference for these antibodies. Anti-PD-L1 antibodiesand uses therefor are described in U.S. Pat. No. 8,552,154, which isincorporated by reference for these antibodies. Anticancer agentcomprising anti-PD-1 antibody or anti-PD-L1 antibody are described inU.S. Pat. No. 8,617,546, which is incorporated by reference for theseantibodies.

In some embodiments, the PDL1 inhibitor comprises an antibody thatspecifically binds PDL1, such as BMS-936559 (Bristol-Myers Squibb) orMPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises anantibody that specifically binds PD1, such as lambrolizumab (Merck),nivolumab (Bristol-Myers Squibb), or MED14736 (AstraZeneca). Humanmonoclonal antibodies to PD-1 and methods for treating cancer usinganti-PD-1 antibodies alone or in combination with otherimmunotherapeutics are described in U.S. Pat. No. 8,008,449, which isincorporated by reference for these antibodies. Anti-PD-L1 antibodiesand uses therefor are described in U.S. Pat. No. 8,552,154, which isincorporated by reference for these antibodies. Anticancer agentcomprising anti-PD-1 antibody or anti-PD-L1 antibody are described inU.S. Pat. No. 8,617,546, which is incorporated by reference for theseantibodies.

The disclosed compositions and methods can be used in combination withother cancer immunotherapies. There are two distinct types ofimmunotherapy: passive immunotherapy uses components of the immunesystem to direct targeted cytotoxic activity against cancer cells,without necessarily initiating an immune response in the patient, whileactive immunotherapy actively triggers an endogenous immune response.Passive strategies include the use of the monoclonal antibodies (mAbs)produced by B cells in response to a specific antigen. The developmentof hybridoma technology in the 1970s and the identification oftumor-specific antigens permitted the pharmaceutical development of mAbsthat could specifically target tumor cells for destruction by the immunesystem. Thus far, mAbs have been the biggest success story forimmunotherapy; the top three best-selling anticancer drugs in 2012 weremAbs. Among them is rituximab (Rituxan, Genentech), which binds to theCD20 protein that is highly expressed on the surface of B cellmalignancies such as non-Hodgkin's lymphoma (NHL). Rituximab is approvedby the FDA for the treatment of NHL and chronic lymphocytic leukemia(CLL) in combination with chemotherapy. Another important mAb istrastuzumab (Herceptin; Genentech), which revolutionized the treatmentof HER2 (human epidermal growth factor receptor 2)-positive breastcancer by targeting the expression of HER2.

Generating optimal “killer” CD8 T cell responses also requires T cellreceptor activation plus co-stimulation, which can be provided throughligation of tumor necrosis factor receptor family members, includingOX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest astreatment with an activating (agonist) anti-OX40 mAb augments T celldifferentiation and cytolytic function leading to enhanced anti-tumorimmunity against a variety of tumors.

In some embodiments, such an additional therapeutic agent may beselected from an antimetabolite, such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine or cladribine.

In some embodiments, such an additional therapeutic agent may beselected from an alkylating agent, such as mechlorethamine, thioepa,chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine(DTIC), procarbazine, mitomycin C, cisplatin and other platinumderivatives, such as carboplatin.

In some embodiments, such an additional therapeutic agent may beselected from an anti-mitotic agent, such as taxanes, for instancedocetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine,vincristine, vinblastine, and vinorelbine.

In some embodiments, such an additional therapeutic agent may beselected from a topoisomerase inhibitor, such as topotecan oririnotecan, or a cytostatic drug, such as etoposide and teniposide.

In some embodiments, such an additional therapeutic agent may beselected from a growth factor inhibitor, such as an inhibitor of ErbBI(EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab,panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinibor erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HER2antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or aninhibitor of both EGFR and HER2, such as lapatinib).

In some embodiments, such an additional therapeutic agent may beselected from a tyrosine kinase inhibitor, such as imatinib (Glivec,Gleevec ST1571) or lapatinib.

Therefore, in some embodiments, a disclosed antibody is used incombination with ofatumumab, zanolimumab, daratumumab, ranibizumab,nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab(Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab(Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and/or rituximab.

In some embodiments, a therapeutic agent for use in combination withdisclosed compositions and methods for treating the disorders asdescribed above may be an anti-cancer cytokine, chemokine, orcombination thereof. Examples of suitable cytokines and growth factorsinclude IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18,IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b),IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.Suitable chemokines may include Glu-Leu-Arg (ELR)-negative chemokinessuch as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C—Cchemokine families. Suitable cytokines include cytokine derivatives,cytokine variants, cytokine fragments, and cytokine fusion proteins.

In some embodiments, a therapeutic agent for use in combination withdisclosed compositions and methods for treating the disorders asdescribed above may be a cell cycle control/apoptosis regulator (or“regulating agent”). A cell cycle control/apoptosis regulator mayinclude molecules that target and modulate cell cycle control/apoptosisregulators such as (i) cdc-25 (such as NSC 663284), (ii)cyclin-dependent kinases that overstimulate the cell cycle (such asflavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01,KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerasemodulators (such as BIBR1532, SOT-095, GRN163 and compositions describedin for instance U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limitingexamples of molecules that interfere with apoptotic pathways includeTNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand(Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-senseBcl-2.

In some embodiments, a therapeutic agent for use in combination withdisclosed compositions and methods for treating the disorders asdescribed above may be a hormonal regulating agent, such as agentsuseful for anti-androgen and anti-estrogen therapy. Examples of suchhormonal regulating agents are tamoxifen, idoxifene, fulvestrant,droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinylestradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), aprogestin (such as such as hydroxyprogesterone caproate,medroxy-progesterone/provera, megestrol acepate/megace), anadrenocorticosteroid (such as hydrocortisone, prednisone), luteinizinghormone-releasing hormone (and analogs thereof and other LHRH agonistssuch as buserelin and goserelin), an aromatase inhibitor (such asanastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or ahormone inhibitor (such as octreotide/sandostatin).

In some embodiments, a therapeutic agent for use in combination withdisclosed compositions and methods for treating the disorders asdescribed above may be an anti-cancer nucleic acid or an anti-cancerinhibitory RNA molecule.

Combined administration, as described above, may be simultaneous,separate, or sequential. For simultaneous administration the agents maybe administered as one composition or as separate compositions, asappropriate.

In some embodiments, the disclosed compositions and methods isadministered in combination with radiotherapy. Radiotherapy may compriseradiation or associated administration of radiopharmaceuticals to apatient is provided. The source of radiation may be either external orinternal to the patient being treated (radiation treatment may, forexample, be in the form of external beam radiation therapy (EBRT) orbrachytherapy (BT)). Radioactive elements that may be used in practicingsuch methods include, e.g., radium, cesium-137, iridium-192,americium-241, gold-198, cobalt-57, copper-67, technetium-99,iodide-123, iodide-131, and indium-111.

In some embodiments, the disclosed compositions and methods isadministered in combination with surgery.

Therapeutic Methods

The disclosed therapeutic compositions may be administered either alone,or as a pharmaceutical composition in combination with diluents and/orwith other components such as IL-2, IL-15, or other cytokines or cellpopulations. Briefly, pharmaceutical compositions may comprise agents orcell populations as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions for use in the disclosedmethods are in some embodimetns formulated for intravenousadministration. Pharmaceutical compositions may be administered in anymanner appropriate treat the cancer. The quantity and frequency ofadministration will be determined by such factors as the condition ofthe patient, and the severity of the patient's disease, althoughappropriate dosages may be determined by clinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the CAR-TIL cells described herein may be administered at adosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kgbody weight, including all integer values within those ranges. CAR-TILcell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimefor a particular patient can readily be determined by one skilled in theart of medicine by monitoring the patient for signs of disease andadjusting the treatment accordingly.

The administration of the disclosed compositions may be carried out inany convenient manner, including by injection, transfusion, orimplantation. The compositions described herein may be administered to apatient subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In some embodiments, the disclosed compositions areadministered to a patient by intradermal or subcutaneous injection. Insome embodiments, the disclosed compositions are administered by i.v.injection. The compositions may also be injected directly into a tumor,lymph node, or site of infection.

In certain embodiments, the disclosed compositions are administered to apatient in conjunction with (e.g., before, simultaneously or following)any number of relevant treatment modalities, including but not limitedto thalidomide, dexamethasone, bortezomib, and lenalidomide. In furtherembodiments, the compositions may be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAM PATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludaribine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, cytokines, andirradiation. In some embodiments, the CAR-TILs are administered to apatient in conjunction with (e.g., before, simultaneously or following)bone marrow transplantation, T cell ablative therapy using eitherchemotherapy agents such as, fludarabine, external-beam radiationtherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.In another embodiment, the cell compositions of the present inventionare administered following B-cell ablative therapy such as agents thatreact with CD20, e.g., Rituxan. For example, in some embodiments,subjects may undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainembodiments, following the transplant, subjects receive an infusion ofthe expanded immune cells of the present invention. In an additionalembodiment, expanded cells are administered before or following surgery.

The disclosed compositions can be used in combination with any compound,moiety or group which has a cytotoxic or cytostatic effect. Drugmoieties include chemotherapeutic agents, which may function asmicrotubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors,or DNA intercalators, and particularly those which are used for cancertherapy.

The disclosed compositions can be used in combination with a checkpointinhibitor. The two known inhibitory checkpoint pathways involvesignaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) andprogrammed-death 1 (PD-1) receptors. These proteins are members of theCD28-B7 family of cosignaling molecules that play important rolesthroughout all stages of T cell function. The PD-1 receptor (also knownas CD279) is expressed on the surface of activated T cells. Its ligands,PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on thesurface of APCs such as dendritic cells or macrophages. PD-L1 is thepredominant ligand, while PD-L2 has a much more restricted expressionpattern. When the ligands bind to PD-1, an inhibitory signal istransmitted into the T cell, which reduces cytokine production andsuppresses T-cell proliferation. Checkpoint inhibitors include, but arenot limited to antibodies that block PD-1 (Nivolumab (BMS-936558 orMDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A,MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX-010),Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3(BMS-986016).

Human monoclonal antibodies to programmed death 1 (PD-1) and methods fortreating cancer using anti-PD-1 antibodies alone or in combination withother immunotherapeutics are described in U.S. Pat. No. 8,008,449, whichis incorporated by reference for these antibodies. Anti-PD-L1 antibodiesand uses therefor are described in U.S. Pat. No. 8,552,154, which isincorporated by reference for these antibodies. Anticancer agentcomprising anti-PD-1 antibody or anti-PD-L1 antibody are described inU.S. Pat. No. 8,617,546, which is incorporated by reference for theseantibodies.

In some embodiments, the PDL1 inhibitor comprises an antibody thatspecifically binds PDL1, such as BMS-936559 (Bristol-Myers Squibb) orMPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises anantibody that specifically binds PD1, such as lambrolizumab (Merck),nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Humanmonoclonal antibodies to PD-1 and methods for treating cancer usinganti-PD-1 antibodies alone or in combination with otherimmunotherapeutics are described in U.S. Pat. No. 8,008,449, which isincorporated by reference for these antibodies. Anti-PD-L1 antibodiesand uses therefor are described in U.S. Pat. No. 8,552,154, which isincorporated by reference for these antibodies. Anticancer agentcomprising anti-PD-1 antibody or anti-PD-L1 antibody are described inU.S. Pat. No. 8,617,546, which is incorporated by reference for theseantibodies.

The disclosed compositions can be used in combination with other cancerimmunotherapies. There are two distinct types of immunotherapy: passiveimmunotherapy uses components of the immune system to direct targetedcytotoxic activity against cancer cells, without necessarily initiatingan immune response in the patient, while active immunotherapy activelytriggers an endogenous immune response. Passive strategies include theuse of the monoclonal antibodies (mAbs) produced by B cells in responseto a specific antigen. The development of hybridoma technology in the1970s and the identification of tumor-specific antigens permitted thepharmaceutical development of mAbs that could specifically target tumorcells for destruction by the immune system. Thus far, mAbs have been thebiggest success story for immunotherapy; the top three best-sellinganticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan,Genentech), which binds to the CD20 protein that is highly expressed onthe surface of B cell malignancies such as non-Hodgkin's lymphoma (NHL).Rituximab is approved by the FDA for the treatment of NHL and chroniclymphocytic leukemia (CLL) in combination with chemotherapy. Anotherimportant mAb is trastuzumab (Herceptin; Genentech), whichrevolutionized the treatment of HER2 (human epidermal growth factorreceptor 2)-positive breast cancer by targeting the expression of HER2.

Generating optimal “killer” CD8 TIL responses may also require T cellreceptor activation plus co-stimulation, which can be provided throughligation of tumor necrosis factor receptor family members, includingOX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest astreatment with an activating (agonist) anti-OX40 mAb augments T celldifferentiation and cytolytic function leading to enhanced anti-tumorimmunity against a variety of tumors.

In some embodiments, such an additional therapeutic agent may beselected from an antimetabolite, such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine or cladribine.

In some embodiments, such an additional therapeutic agent may beselected from an alkylating agent, such as mechlorethamine, thioepa,chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine(DTIC), procarbazine, mitomycin C, cisplatin and other platinumderivatives, such as carboplatin.

In some embodiments, such an additional therapeutic agent may beselected from an anti-mitotic agent, such as taxanes, for instancedocetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine,vincristine, vinblastine, and vinorelbine.

In some embodiments, such an additional therapeutic agent may beselected from a topoisomerase inhibitor, such as topotecan oririnotecan, or a cytostatic drug, such as etoposide and teniposide.

In some embodiments, such an additional therapeutic agent may beselected from a growth factor inhibitor, such as an inhibitor of ErbBI(EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab,panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinibor erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HER2antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or aninhibitor of both EGFR and HER2, such as lapatinib).

In some embodiments, such an additional therapeutic agent may beselected from a tyrosine kinase inhibitor, such as imatinib (Glivec,Gleevec ST1571) or lapatinib.

Therefore, in some embodiments, a disclosed antibody is used incombination with ofatumumab, zanolimumab, daratumumab, ranibizumab,nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab(Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab(Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and/or rituximab.

In some embodiments, a therapeutic agent for use in combination withcompositions for treating the disorders as described above may be ananti-cancer cytokine, chemokine, or combination thereof. Examples ofsuitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6,IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a,IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN, GM-CSF, CD40L, Flt3ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines mayinclude Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG,and SDF-la from the human CXC and C—C chemokine families. Suitablecytokines include cytokine derivatives, cytokine variants, cytokinefragments, and cytokine fusion proteins.

In some embodiments, a therapeutic agent for use in combination with acomposition for treating cancers as described above may be a cell cyclecontrol/apoptosis regulator (or “regulating agent”). A cell cyclecontrol/apoptosis regulator may include molecules that target andmodulate cell cycle control/apoptosis regulators such as (i) cdc-25(such as NSC 663284), (ii) cyclin-dependent kinases that overstimulatethe cell cycle (such as flavopiridol (L868275, HMR1275),7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine(R-roscovitine, CYC202)), and (iii) telomerase modulators (such asBIBR1532, SOT-095, GRN163 and compositions described in for instanceU.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limiting examples ofmolecules that interfere with apoptotic pathways include TNF-relatedapoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L),antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

In some embodiments, a therapeutic agent for use in combination withcompositions for treating cancers as described above may be a hormonalregulating agent, such as agents useful for anti-androgen andanti-estrogen therapy. Examples of such hormonal regulating agents aretamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene,diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such asflutaminde/eulexin), a progestin (such as such as hydroxyprogesteronecaproate, medroxy-progesterone/provera, megestrol acepate/megace), anadrenocorticosteroid (such as hydrocortisone, prednisone), luteinizinghormone-releasing hormone (and analogs thereof and other LHRH agonistssuch as buserelin and goserelin), an aromatase inhibitor (such asanastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or ahormone inhibitor (such as octreotide/sandostatin).

In some embodiments, a therapeutic agent for use in combination withcompositions for treating the cancers as described above may be ananti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

Combined administration, as described above, may be simultaneous,separate, or sequential. For simultaneous administration the agents maybe administered as one composition or as separate compositions, asappropriate.

In some embodiments, the disclosed compositions are administered incombination with radiotherapy. Radiotherapy may comprise radiation orassociated administration of radiopharmaceuticals to a patient isprovided. The source of radiation may be either external or internal tothe patient being treated (radiation treatment may, for example, be inthe form of external beam radiation therapy (EBRT) or brachytherapy(BT)). Radioactive elements that may be used in practicing such methodsinclude, e.g., radium, cesium-137, iridium-192, americium-241, gold-198,cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, andindium-111.

In some embodiments, the disclosed compositions are administered incombination with surgery.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1 Background

Stimulator of interferon genes (STING) is an endoplasmicreticulum-resident signaling molecule. It is responsible for controllingthe transcription of several host defense genes, including type I IFNsand pro-inflammatory cytokines, in response to recognition of cytosolicDNA species or cyclic dinucleotides. Recent studies have indicated thatSTING signaling is the major innate immune pathway involved in thegeneration of a spontaneous antitumor T cell response. Mice lackingSTING cannot generate efficient antitumor T cell responses and rejectmelanoma tumor growth. Based on this finding, many STING agonists havebeen developed to utilize STING activation as a cancer therapy. Suchagonists have been found to be experimentally useful in inducing robusttumor control through the host immune cell activation. While STINGactivation has been extensively investigated in antigen presenting cells(in particular dendritic cells), little is known regarding itsactivation in tumor cells.

Methods

To gain more insights into the role of STING signaling in melanoma,first we have explored the expression of STING and cGAS (cyclic GMP-AMPsynthase) in a panel of human melanoma cell lines by immunoblot. Next,we have studied the functional STING signaling activation in STINGpositive melanoma cells following their stimulation with a known STINGagonist 2′3′-cGAMP by measuring the induction of CXCL-10 and IFN-□ usingELISA.

To determine if epigenetic processes such as hypermethylation areinvolved in the suppression of STING expression and its functionalsignaling, we have treated melanoma cells lacking STING expression withthe demethylating agent 5-aza-2′-deoxycytidine (5AZADC) and evaluatedinduction of STING expression by immunoblot.

To study the role of STING signaling in immunogenicity of melanoma, wehave set up co-cultures of expanded human melanoma tumor infiltratinglymphocytes (TILs) with their HLA-matched melanoma cell lines in thepresence of the STING agonist 2′3′-cGAMP. We have assessed thesubsequent immunogenicity by IFN-γ release and 51Cr release cytotoxicityassays.

Results

Examining STING and cGAS expression by immunoblot showed that there is adiverse STING/cGAS expression status among human melanoma cell lines.STING expression was not detectable in 11 of 18 human melanoma celllines. Also, STING activation in majority of human melanoma cell lineswas found to be defective.

Partial induction of STING expression in 5AZADC-treated melanoma celllines lacking STING and induction of CXCL-10 following their stimulationwith the STING agonist suggested DNA hypermethylation may be involved inthe suppression of STING signaling.

Activation of STING pathway in human melanoma cell lines when culturedwith their HLA-matched TILs resulted in increased IFN-□ secretionsuggesting the presence of a STING-induced immunogenicity. 51Cr releasecytotoxicity assay further confirmed that STING activation in humanmelanoma cells augments their cytotoxic T lymphocyte-mediated lysis.Accordingly, we have found that STING activation in melanoma cellsinduces increased surface expression of MHC class I which could allowthem to be more effectively recognized by cytotoxic T cells.

There is a Diverse STING/cGAS Expression Status Among Melanoma CellLines.

STING expression is suppressed and dsDNA-induced innate immuneactivation is impaired in majority of human melanoma cell lines. FIGS.1A, 1B, and Table 1 show STING and cGAS expression in melanoma celllines.

TABLE 1 STING and cGAS expression in human melanoma cell lines Cell LineSTING cGAS BRAF WM 35 − + V600E 888 − − V600E SBCL2 − Low V600E A375 Low− V600E 1366  + − V600E WM 39 + − V600E 526 Low − V600E 1205  − + V600EWM 3130 − − ? WM 982 B − + ? 239 − − V600D WM 9 + + V600D WM 2032 − +−V600E WM 858 − + V600D WM 3629 + + D549G het 266-4 − + V600D WM 164 + +V600E WM 1361 A − + WT

STING Signaling Pathway is Defective in Majority of Human Melanoma CellLines.

FIG. 2 illustrates the activation of STING pathway in NK92 cells(positive control) with 2′3′-cGAMP.

FIG. 3 is a bar graph showing induction of CXCL-10 expression in NK92cells after 4 h treatment with 2′3-cGAMP.

Table 2 shows the activation of STING pathway in NK92 cells (positivecontrol) with 2′3′-cGAMP.

TABLE 2 Activation of STING pathway in human melanoma cell lines with2′3′-cGAMP Cell Line STING cGAS WM 164 + + WM 9 + + WM 39 + − A375 low −1366 + − 2032 − +

FIGS. 4A to 4C show analysis of STING signaling activation in humanmelanoma cell lines.

FIGS. 5A and 5B show expression of CXCL-10 and IFN-b in WM9 and WM39cell lines after 4 h & 24 h treatment with 2′3′ cGAMP.

FIG. 6 is a bar graph showing expression of CXCL-10 in STING positivemelanoma cell lines after 4 h & 24 h treatment with 2′3-cGAMP.

DNA Hypermethylation May be Involved in the Suppression of STINGSignaling in Melanoma

FIGS. 7A to 7E show DNA demethylation partially recapitulated STING andcGAS expression in human melanoma cell lines.

FIGS. 8A to 8D show reconstitution of STING expression in 5AZADC-treatedmelanoma cells and STING-dependent CXCL-10 induction following2′3′-cGAMP stimulation.

FIGS. 9A to 9F show CXCL-10 induction in 5AZADC-treated melanoma cellsfollowing 2′3′-cGAMP stimulation.

In summary, CXCL-10 induction in 5AZADC-treated melanoma cell linesfollowing stimulation with the STING agonist suggests DNAhypermethylation may be involved in suppressing STING signaling.

STING Signaling Impact in Immunogenicity of Melanoma

A co-culture assay was performed that involved melanoma cells treatedwith or without 2′3′ cGAMP (WM39 (HLA A01/02)) with melanoma TILs(V40195 (A2), 40019 (A2)). Specifically, WM39 cells were plated at 2×10⁶cells/ml and 50 μl/well (1×10⁶ cells/well) with W6/32 Ab (HLA-ABCantibody). Next, 2′3′-cGAMP was added to WM39 cells at 50 μl/well. Thiswas incubated for 1 hour. The TILs were then added and incubated foranother hour. The co-culture supernatants were then collected andevaluated for IFN-γ by ELISA.

FIG. 10 is a bar graph showing activation of STING signaling in MART-1pulsed WM39 melanoma cells results in increased IFN-γ secretion whencultured with V40195 TIL.

FIGS. 11A to 11I are bar graph showing IFN-γ, CXCL-10 & IFN-β expressionin co-cultures of MART-1 pulsed WM39 with HLA-matched TILs.

FIGS. 12A to 12I are bar graph showing IFN-g, CXCL-10 & IFN-b expressionin co-cultures of WM39, MART-1 pulsed WM39 or WM3629 with V40195 TIL.

FIGS. 13A and 13B show activation of STING pathway in human melanomasimproves their cytotoxic T lymphocyte-mediated lysis.

FIG. 14 shows MHC-I/HLA-A.B.C expression in melanoma cell lines aftertreatment with 2′3′ cGAMP. STING activation in human melanoma cell linesinduces upregulation of MHC 1 (HLA-A.B.C).

In summary, activation of STING pathway in human melanoma cell linesimproves their immunogenicity when cultured with HLA-matched TILs;improves their cytotoxic T lymphocyte-mediated lysis; and inducesupregulation of MHC 1 (HLA-A.B.C).

Example 2: STING Signaling can Enhance Melanoma Antigenicity

Methods

STING and cGAS expression were examined in a panel of human melanomacell lines by immunoblot. Functional STING signaling activation wasexamined in STING-positive melanoma cell lines upon stimulation with theagonist 2′3′-cGAMP by measuring the induction of CXCL-10 and IFN-β. Todetermine if hypermethylation was involved in the suppression of STINGexpression and signaling where gene mutations were absent, melanomacells lacking STING expression were treated with 5-aza-2′-deoxycytidine(5AZADC). To study the role of STING signaling on antigenicity ofmelanoma, expanded human tumor infiltrating lymphocytes (TIL) wereco-cultured with their HLA-matched melanoma cell lines in the presenceor absence of 2′3′-cGAMP agonist. TIL production of IFN-γ and ⁵¹Crrelease was assessed for cytotoxicity.

Results

Immunoblot analysis revealed a diverse STING/cGAS expression status inhuman melanoma cell lines. STING expression was not detected in 11 of 18of them. Induction of STING expression in 5AZADC-treated melanoma celllines lacking STING and production of CXCL-10 following theirstimulation with the STING agonist suggested DNA hypermethylationinvolvement in cases where STING gene mutations were absent. AmongSTING-positive cell lines, two responded strongly to STING signalingactivation with 2′3′-cGAMP. Activation of the STING pathway in thesecell lines when cultured with their HLA-matched TILs resulted in up to a15-fold increase in IFN-γ secretion (p<0.01) as well augmentation of TILcytotoxicity by >2-fold (p<0.05). In addition, STING activation couldinduce enhanced surface expression of MHC class I in human melanoma celllines leading to more effective tumor antigen recognition by TIL.

CONCLUSIONS

Direct activation of the STING pathway in human melanoma cell lines canresult in improved antigenicity.

Example 3: Tumor Cell-Intrinsic STING Signaling Impacts Antigenicity ofMelanoma and can Promote Antitumor T-Cell Activity

Methods

Preparation of TIL

Melanoma TIL were established as described previously (Pilon-Thomas S,et al. J Immunother. 2012 35:615-20). Briefly, melanomas were mincedinto 1-2 mm³ fragments and plated in 24-well plates with 2 mL TILculture medium (TIL-CM) containing 6000 IU/mL IL-2 (Proleukin) per well.The TIL-CM consisted of RPMI 1640, 2.05 mM L-glutamine (HyClone, ThermoFisher Scientific), 10% heat-inactivated human AB serum (OmegaScientific), 55 μM 2-mercaptoethanol (Invitrogen), 50 μg/mL gentamicin(Invitogen), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 mMHEPES Buffer (Mediatech). Half of the medium was replaced every 2 to 3days or wells were split when 90% confluent. TIL were expanded for 3-5weeks. HLA typing of TIL was performed by the HLA Laboratory (AmericanRed Cross, Dedham, Mass.). TIL 195, TIL 19 and TIL 123 were HLA-A typedas A02, A02/26 and A02/11, respectively.

Melanoma Cell Lines

Human melanoma cell lines 1205Lu, A375, SBCL2, WM9, WM35, WM39, WM164,WM858, WM1361A, WM1366, WM2032, WM3130, WM3629, 526-MEL and 888-MEL weremaintained as monolayers in complete medium consisting of RPMI 1640supplemented with 10% heat-inactivated FBS and antibiotics. HLA typingof melanoma cell lines was performed by the HLA Laboratory (American RedCross, Dedham, Mass.). WM39, WM3629 and 526-MEL were HLA-A typed asA01/02, A02/30 and A02/03, respectively.

Knockdown of STING in WM39 cells was achieved using lentiviral particlescarrying a target gene sequence for human STING (TMEM173) or scrambledcontrol (Origene Technologies). Transduced cells were selected byaddition of puromycin (0.5 μg/ml) to the medium 24 h after infection.

STING Agonist Stimulation

Human melanoma cell lines (4×10⁵ cells/well in 24-well plates) werestimulated with 2′3′-cGAMP (10 μg/ml) in the presence of Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. After 4or 24 hours of incubation at 37° C. in a humidified CO₂ incubator, thesupernatants were collected for detection of CXCL10 and IFN-β releaseusing enzyme-linked immunosorbent assays (Quantikine ELISA Kit, R&DSystems), and cells were scraped, washed and lysed for assessment ofIRF3 phosphorylation by immunoblot.

Immunoblot Analysis

Proteins were extracted with RIPA buffer (ThermoFisher Scientific)containing protease inhibitors (Thermo Scientific). Protein extractsfrom NK92, a natural killer cell line, was used as a positive controlfor the expression of STING and cGAS (Souza-Fonseca-Guimaraes F, et al.J Biol Chem. 2013 288:10715-21). Equal amounts of proteins were resolvedon SDS-PAGE gels (Bio-Rad) and transferred to polyvinylidene fluoride(PVDF) membranes (Bio-Rad). After blocking with 5% non-fat dry milk,membranes were incubated with antibodies specific for STING, cGAS,p-IRF3, IRF3 (all from Cell Signaling) and β-actin (Sigma Aldrich).Following incubation with appropriate secondary antibodies, bands werevisualized using an enhanced chemiluminescence detection system.

Co-Culture Assay

1×10⁵ of melanoma cells were cultured with TIL at a 1:1 ratio with orwithout 2′3′-cGAMP (10 μg/ml) in 96-well round-bottom plates. After 24hours of incubation at 37° C. in a humidified CO₂ incubator, thesupernatant was harvested for detection of IFN-γ release usingenzyme-linked immunosorbent assay (Human IFN-γ Quantikine ELISA Kit, R&DSystems). For the MHC class I blocking assay, melanoma cells wereincubated with W6/32 (anti-HLA-A,B,C monoclonal antibody, Biolegend) ata final concentration of 50 μg/mL for 1 hour at 37° C. prior to theaddition of TIL.

⁵¹Cr Release Assay

Lysis of melanoma cell targets by their HLA-matched TIL cultures wasmeasured in a standard ⁵¹Cr release assay, as described previously (ZhuG, et al. Front Immunol. 2018 9:1609). Briefly, 1×10⁶ melanoma cellswere labeled with 100 μCi of ⁵¹Cr (Amersham Corp) for 2 h at 37° C.Following three washes with HBSS, labeled target cells were resuspendedin TIL CM with or without 2′3′-cGAMP (10 μg/ml) at a concentration of5×10⁴ tumor cells/ml and added to the effector cells at differenteffector-to-target cell ratios in a 96-well plate and incubated at 37°C. In addition, two control conditions were included in this assay: aminimum release control containing just the target cells and a maximumrelease control in which target cells were lysed by TritonX-100. After 4hours, supernatant was harvested and measured in Trilux (PerkinElmer).Each point represented the average of quadruplicate wells and percentageof specific lysis was calculated by: (experimental release−minimumrelease)/(maximum release−minimum release)×100. Lytic units werecalculated as the number of effector cells required to produce 20% lysisof 5×10³ target cells expressed as the inverse and normalized to 1×10⁶cells (Bryant J, et al. J Immunol Methods. 1992 146:91-103).

Flow Cytometry

Flow cytometry was performed using HLA-A.B.C-PE antibody (1:100,Biolegend, clone W6/32). DAPI (Sigma-Aldrich) was used as a viabilitydye. Sample acquisition was performed on an LSR II flow cytometer (BDBiosciences), and the data were analyzed using FlowJo software (TreeStar).

Statistical Methods

Statistical analyses were performed using GraphPad Prism7 software. Alldata are presented as mean±SEM. Means for all data were compared byunpaired Student's t-test or one-way ANOVA. P-values of <0.05 wereconsidered statistically significant. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

Results

Identification of Melanoma Cell Lines with Intact STING Signaling

To identify melanoma cell lines with intact STING signaling, expressionof STING and cGAS was first evaluated in a panel of human melanoma celllines by immunoblot (FIG. 15A). There were varying expression levels ofSTING and cGAS among these cell lines consistent with a previous report(Xia T, et al. Cancer research. 2016 76:6747-59). STING was notdetectable in 9 of 16 melanoma cell lines (WM35, 888-MEL, SBCL2, 1205Lu,WM2032, WM858, WM266-4, WM1361A and WM3130). We identified A375, WM1366,WM39, WM9, WM3629 and WM164 as STING-positive cell lines (FIG. 15B).cGAS was detected in 10 of 16 cell lines (FIG. 15C), and only 3 celllines (WM9, WM3629 and WM164) expressed both cGAS and STING.

Next investigated was the functional STING signaling activation bystimulating melanoma cell lines with the STING agonist 2′3′-cGAMP. Asthis agonist activates STING signaling in a cGAS-independent manner, 5STING-positive cell lines (WM164, WM9, WM39, A375 and WM1366) and 1STING-negative cell line (WM2032) were. Immunoblot analysis wasperformed on cell extracts after stimulation with 2′3′-cGAMP to assessphosphorylation of the transcription factor IRF3, which is a criticaldownstream regulatory element for STING-dependent type I IFN induction(Tanaka Y, et al. Science signaling. 2012 5:ra20). phosphorylation ofIRF3 in 4 of 5 STING-positive cell lines was observed following theirstimulation with 2′3′-cGAMP (FIGS. 15D and 15E). As expected,phosphorylation of IRF3 was not detected for the 2′3′-cGAMP stimulatedWM2032 (STING-negative) cell line. STING-dependent CXCL10 and IFN-βinduction was also determined in cell culture supernatants of theindicated melanoma cell lines following stimulation with 2′3′-cGAMP(FIGS. 15F and 15G). Among these cell lines, WM9 and WM39 induceddetectable levels of CXCL10 and IFN-β in response to stimulation with2′3′-cGAMP.

Activation of STING Signaling Results in Enhanced Antigenicity of HumanMelanoma Cell Lines

To study the role of STING signaling in antigenicity of melanoma, WM39(HLA-A2) was initially selected, as this cell line responded strongly toSTING signaling activation with 2′3′-cGAMP, and it was used inco-cultures of HLA-A2-restricted human melanoma TIL (TIL 195) in thepresence or absence of the STING agonist. Experimental conditions inwhich WM39 cells were pre-incubated with an MHC class I blocking Ab(w6/32) were also included to determine whether IFN-γ release wasmediated by CD8+ TIL TCR engagement with peptide/MHC class I.

The antigenicity by IFN-γ release was assessed, demonstrating that whenco-cultures were performed with 2′3′-cGAMP-treatment, there wassignificantly enhanced IFN-γ secretion by HLA-matched TIL 195 (FIG. 2A).Blockade of IFN-γ release was also observed in the presence of the MHCclass I blocking Ab (w6/32), which confirmed MHC class I-mediated CD8⁺reactivity.

CXCL10 and IFN-β (FIG. 12) levels were also measured in co-culturesupernatants to confirm 2′3′-cGAMP-triggered activation of STINGsignaling. Although there was CXCL10 induction in the co-culture groupwithout 2′3′-cGAMP, this effect was related to STING-independent andIFN-γ-mediated induction of CXCL10 in WM39 cells (Peng W, et al. Cancerresearch. 2012 72:5209-18). Induction of IFN-β in co-culture groups with2′3′-cGAMP confirmed activation of STING signaling. IFN-β induction wasnot observed for the control group containing TIL with 2′3′-cGAMP whichsuggests tumor cells are the main source of IFN-β expression in theco-culture group in response to stimulation with the agonist. Takentogether, these data indicate that activation of STING signaling plays anotable role in enhancing antigenicity of WM39 cells.

To further investigate the impact of STING activation onantigen-presentation and immune T cell activity, WM39 cells were pulsedwith MART-1 (a known melanoma specific peptide recognized byHLA-A2-restricted TIL (Kawakami Y, et al. J Exp Med. 1994 180:347-52)),and co-cultured them with TIL 195 in the presence or absence of theSTING agonist 2′3′-cGAMP (FIG. 12). Similarly, there was a significantlyincreased (p<0.001) secretion of IFN-γ by TIL 195 in the2′3′-cGAMP-treated co-culture group. Consistent with the WM39co-cultures, there were similar patterns of CXCL10 and IFN-β (FIG. 12)induction for MART-1-pulsed WM39 co-cultures, which confirmed activationof STING signaling in 2′3′-cGAMP-treated groups.

MART-1 pulsed WM39 cells were also co-cultured with two additionalHLA-A2 TIL. Similarly, there were higher levels of IFN-γ production(p<0.01) by both TIL samples in 2′3′-cGAMP-treated co-cultures comparedto the untreated co-cultures. Furthermore, WM3629 (HLA-A2) melanoma cellline was tested that in earlier experiments responded to 2′3′-cGAMPstimulation but to a lesser extent than WM39, and used it in co-cultureswith TIL 195 in the presence or absence of the STING agonist (FIG. 12).Stimulation with 2′3′-cGAMP similarly resulted in increased (p<0.01)IFN-γ release in WM3629/TIL 195 co-cultures, indicating that theenhancing effect of 2′3′-cGAMP was not restricted to the WM39 melanomacell line.

There was a notable decrease in the expression of CXCL10 in theagonist-treated co-cultures compared to the untreated co-cultures (FIGS.12A-12I). As tumor cells are the primary source of CXCL10 expression,this is likely an indication that more tumor cells were lysed by TIL inthe agonist-treated groups compared to the untreated controls.

STING Signaling is Required in Melanoma Cells for Agonist-InducedImproved Antigenicity

Given the pronounced impact of the STING agonist on IFN-γ release inmelanoma/TIL cocultures, the next goal was to determine whether this wasdue to the direct activation of STING signaling in melanoma cells. Toaddress this possibility, the 526-MEL (HLA-A2) melanoma cell line thatdid not respond to 2′3′-cGAMP stimulation (FIG. 16A-16C) was co-culturedwith two HLAA2-restricted TIL (TIL 19 and TIL 195) in the presence orabsence of the STING agonist and in parallel co-cultures we used WM39cells with the same TIL samples (FIGS. 16D and 16E). 526-MEL stimulatedmuch higher amounts of IFN-γ release from TIL 19 compared to WM39 cells.However, in contrast to WM39/TIL 19 co-cultures for which stimulationwith 2′3′-cGAMP resulted in 24-fold higher (p<0.001) IFN-γ release thanthe untreated group, agonist treatment did not induce any increasedIFN-γ secretion for 526-MEL/TIL 19 co-culture (FIG. 16D). Similarly, noincrease in IFN-γ release was observed for the 526-MEL/TIL 195co-cultures in the presence of the agonist (FIG. 16E), supporting thatSTING agonist-mediated enhanced antigenicity is driven by activation ofSTING signaling in melanoma cells.

Activation of STING Pathway in Human Melanoma Cell Lines ImprovesCytotoxic T Lymphocyte-Mediated Lysis

⁵¹Cr release cytotoxicity assays were next performed using WM39, MART-1pulsed WM39, WM3629, and 526-MEL as target cells and TIL 195 as effectorcells in the presence or absence of 2′3′-cGAMP, to determine cytolyticactivity of TIL against melanoma cells stimulated with the agonist.Similar to the finding of increased TIL production of IFN-γ, STINGactivation in both WM39 and MART-1 pulsed WM39 cells resulted in markedincreases in their lysis by TIL 195 (>2-fold, p<0.05) (FIGS. 17A and17B). Blocking MHC class I in WM39 targets effectively inhibitedspecific TIL lysis in agonist treated groups (FIG. 17C), indicating thatthe enhanced cytotoxic activity in response to activation of STINGsignaling was driven by MHC class I restricted TIL. To further confirmthat this effect was mediated by cytolytic activity of TIL per se andnot by the STING agonist, two control groups in which WM39 target cellswere incubated with or without 2′3′-cGAMP in the absence of TIL wereincluded. No significant difference of cytotoxicity in these two groupswas found, which argued that stimulation with the STING agonist alonedid not result in any major cytotoxicity. Increased (p<0.05)cytotoxicity in 2′3′-cGAMP-treated WM3629 cells was observed compared tothe controls (FIG. 17D). However, 2′3′-cGAMP stimulation of 526-MELtargets, which were defective in STING signaling, did not alter theirspecific lysis by TIL 195. To better compare the cytolytic activity ofTIL against different agonist-treated and untreated melanoma targets, wecalculated lytic units (FIG. 17E). There was more than a 10-foldincrease in lytic potential of TIL 195 against 2′3′-cGAMP-treated WM39and MART-1-pulsed WM39 cells compared to their controls. Similarly,activation of STING in WM3629 cells resulted in in greater lysis by TIL195. In contrast, 2′3′-cGAMP-treated 526-MEL cells did not induce anyincrease in lysis by TIL 195.

STING Activation in Human Melanoma Cell Lines Induces Up-Regulation ofMHC Class I

Following the observation of enhanced antigenicity of human melanomacell lines triggered by agonist-induced activation of STING signaling,the expression of MHC class I on 2′3′-cGAMP-stimulated melanoma cellswas next examined. Surface expression of MHC class I in all four celllines with functional STING signaling (WM9, WM3629, A375 and WM39) wassignificantly increased (p<0.01, fold change >1.6) following thestimulation with the STING agonist (FIGS. 18A and 18B). In contrast,there were no significant changes in the expression of MHC class I in1205Lu, WM266-4, WM2032 and 526-MEL cell lines with an impaired STINGsignaling following their exposure to the agonist. Together, theseresults indicated that activation of STING signaling could induceup-regulation of MHC class I in a subset of melanoma cell lines, leadingto more effective immune recognition and antigen presentation to TIL.

Knockdown of STING Blocks Agonist-Induced Upregulation of MHC Class I inMelanoma Cells

STING expression in WM39 cells was next stably knocked down withlentivirus-based short hairpin RNA targeting STING (sh-STING). We usedWM39 cells expressing a non-targeting hairpin as control cells(sh-control). Immunoblot analysis indicated complete deletion of STINGin WM39 cells transduced with sh-STING (FIG. 19A). Knockdown of STINGblocked phosphorylation of IRF3 and agonist-induced induction of CXCL10and IFN-β in WM39 cells in response to stimulation with 2′3′-cGAMP (FIG.19B-19D). Unlike WM39 and sh-control for which stimulation with theagonist resulted in more than 2.5-fold higher surface expression of MHCclass I (p<0.01), stimulation with the agonist did not cause anyincrease in MHC class I for sh-STING cells (FIGS. 19E and 19F)demonstrating that the upregulation of MHC class I in response tostimulation with the agonist occurs through activation of STINGsignaling.

STING is Essential for Agonist-Induced Enhanced Antigenicity in MelanomaCells

To further confirm the role of STING signaling in enhancing antigenicityof melanoma, sh-STING, sh-control and non-transfected WM39 cells wereused in co-cultures with TIL 195 in the presence and absence of2′3′-cGAMP. In contrast to WM39/TIL 195 and sh-control/TIL 195co-cultures for which stimulation with the agonist resulted in more than25-fold higher IFN-γ release (P<0.05), no increase in IFN-γ inductionwas observed in sh-STING/TIL 195 co-cultures in the presence of theagonist (FIG. 7A). ⁵¹Cr cytotoxicity assays were also performed usingsh-STING, sh-control and WM39 as target cells and TIL 195 as effectorcells at different effector to target ratios with or without 2′3′-cGAMP.While there was increased cytolytic activity with TIL 195 against bothWM39 and sh-control in the presence of the agonist, inhibition of STINGsignaling in sh-STING led to complete blockade of this response (FIG.7B), as reflected by the corresponding lytic units (FIG. 7C).

DISCUSSION

Immunotherapies including adoptive cell transfer of tumor infiltratinglymphocytes and immune checkpoint inhibitor antibodies have shownefficacy in patients with metastatic melanoma (Rosenberg S A, et al.Science. 2015 348:62-8; Pilon-Thomas S, et al. J Immunother. 201235:615-20; Postow M A, et al. N Engl J Med. 2015 372:2006-17). However,there remains a notable subset of melanoma patients treated withimmune-based therapies that does not achieve clinical benefit (26-28).Therefore, understanding the mechanisms underlying both successful andfailed immune responses has important implications for improvingimmunotherapeutic approaches.

As TIL-based immunotherapies have been developed on the basis ofexpanding tumor-reactive T cells from the tumor microenvironment, it isevident that a spontaneous adaptive immunity exists within tumors,although in a dysfunctional state (Topalian S, et al. J Immunol. 1989142:3714-25; Robbins P F, et al. Nat Med. 2013 19:747-52). Moreover, apre-existing CD8+ T cell infiltrate within tumors has been stronglyassociated with clinical response to checkpoint blockade immunotherapiesin melanoma patients (Tumeh P C, et al. Nature. 2014 515:568-71),indicating the prognostic significance of endogenous T cell responses.These observations have led to an important question regarding how theinnate immune system could detect cancer and initiate a spontaneousadaptive T cell response against tumor antigens without the presence ofinfectious pathogens (Corrales L, et al. J Immunol. 2013 190:5216-25).

Studies using different gene-targeted mouse models deficient in specificinnate immune pathways have identified STING pathway to be the majorinnate immune sensing mechanism for the detection of immunogenic tumorsand initiation of a spontaneous T cell response (Woo S-R, et al.Immunity. 2014 41:830-42). Based on this finding, multiple studies havebeen conducted to evaluate whether direct activation of STING signalingusing pharmacologic STING agonists could be used in facilitatingantitumor immune responses in mouse models (Corrales L, et al. Cellreports. 2015 11:1018-30; Conlon J, et al. The Journal of Immunology.2013 190:5216-25; Fu J, et al. Sci Transl Med. 2015 7:283ra52). Whilethey all found enhanced therapeutic activity by intratumoraladministration of STING agonists, mechanistic details regarding howdirect activation of STING signaling could potentiate antitumor immunityremain largely unknown. In fact, it is currently unclear how STINGagonists could impact cell types other than APCs in a tumormicroenvironment, in particular tumor cells.

Although evidence suggests STING signaling is frequently impaired inhuman melanoma cells (Xia T, et al. Cancer research. 2016 76:6747-59),there remains a subset of melanomas with STING expression for which thefunctional significance of STING activation has not been well explored.In this study, it is shown that many melanoma cell lines have lost theexpression of STING and are therefore defective in responding tostimulation with the STING agonist 2′3′-cGAMP. Also shown is impairedfunctional responses to stimulation with the STING agonist in somemelanoma cells that expressed STING, suggesting that STING signaling canbe inhibited not only by suppression of STING/cGAS expression but alsothrough other molecular mechanisms that remain to be determined (Xia T,et al. Cancer research. 2016 76:6747-59; Chen Q, et al. Nat Immunol.2016 17:1142-9). Interestingly, stimulation with the 2′3′-cGAMP agonistcan induce potent STING activation in a subset of human melanoma celllines leading to downstream production of IFN-β and CXCL10. Suchagonist-induced activation of the STING pathway can increaseantigenicity of melanoma cells through the augmentation of MHC class Iexpression and result in better tumor-antigen recognition by immune Tcells.

Down-regulation of MHC class I is one of the major mechanisms used bytumor cells to evade host immune recognition (Seliger B, et al. ImmunolToday. 2000 21:455-64; Hicklin D J, et al. J Clin Invest. 1998101:2720-9). Loss or down-regulation of MHC class I was associated withsignificantly lower levels of tumor infiltrating lymphocytes and poorclinical outcomes in patients with metastatic melanoma. Suchcorrelations were found in melanoma cell lines derived from bothrecurrent metastases in patients who had initially experienced clinicalresponses to TIL-based therapies (Restifo N P, et al. J Natl CancerInst. 1996 88:100-8; Garrido F, et al. Curr Opin Immunol. 2016 39:44-51;Chang C-C, et al. J Immunol. 2005 174:1462-71) or from previouslyuntreated melanoma patients that showed resistance to anti-CTLA-4therapy later (Rodig S J, et al. Sci Transl Med. 2018 10:eaar3342). Incontrast, tumor regression was correlated with positive tumor MHC classI expression, highlighting the functional significance of antigenpresentation by tumor cells in the initiation of successful anti-tumorresponses (Carretero R, et al. Int J Cancer. 2012 131:387-95).

Although the molecular mechanism(s) underlying STING agonist-inducedup-regulation of MHC class I remains undefined, it depends on activationof STING signaling, as up-regulation of MHC class I was not observed inmelanoma cell lines with defective STING signaling following theirstimulation with 2′3′-cGAMP. Also, findings suggest that STINGagonist-mediated up-regulation of MHC class I occurs through type I IFNdependent mechanisms, as this effect was found in tumor cells stimulatedwith the agonist in the absence of TIL and IFN-γ. Taken together, datasupport the concept that agonist-induced intact activation of STINGsignaling in melanomas could be considered as a therapeutic interventionto restore MHC class I surface expression and subsequently to enhancetumor antigen recognition and tumor cell destruction by immune T cells.

Activation of STING signaling in melanoma cell lines results indownstream induction of IFN-β. STING-mediated IFN-β induction indendritic cells has been found to be essential for their activation andthe cross-priming of cytotoxic T cells (Woo S-R, et al. Immunity. 201441:830-42). Similarly, recent work has shown that initiation of a robustadaptive immune response to radiation therapy requires STING-mediatedIFN-β induction in dendritic cells (Deng L, et al. Immunity. 201441:843-52).

Although the contribution of IFN-β produced by tumor cells in responseto STING agonists in modulating interactions between tumor cells and theimmune system and subsequent initiation of tumor-specific T-cellresponses had not been previously explored, there was evidence thatagonist-mediated induction of IFN-β in melanoma cell lines whenco-cultured with their HLA-matched TIL strongly correlates withincreased TIL production of IFN-γ and T lymphocyte-mediatedcytotoxicity. In addition, IFN-β induction in tumor cells has beenpreviously reported to initiate a series of orchestrated eventsinvolving autocrine and paracrine signals resulting in inducing multipleeffects on both tumor cells and antigen presenting cells, includingregulation of antigen processing, peptide transfer, peptide-loadingcomplex, MHC class I expression, or downstream induction of othercytokines and/or chemokines such as CXCL10 (Diamond M S, et al. J ExpMed. 2011 208:1989-2003; Sistigu A, et al. Nat Med. 2014 20:1301-9).Therefore, it may be of benefit to determine the functional contributionof IFN-β induced by tumor cells in response to the STING agonist ininitiation of the antitumor immunity.

As one of the major characteristic chemokines of type I IFN immuneresponse and STING signaling (Barber G N. Trends Immunol. 2014 35:88-93;Zitvogel L, et al. Nat Rev Immunol. 2015 15:405-14), there wasdownstream induction of CXCL10 in melanoma cell lines with intact STINGsignaling following their stimulation with 2′3′-cGAMP. Along with CXCL9,another CXCR3-binding chemokine, CXCL10 has been identified as thedominant mediator for the recruitment of CXCR3+ tumor-specific Tlymphocytes into the tumors and its intratumoral expression correlatedwith favorable clinical outcomes in patients with melanoma andcolorectal cancer (Mlecnik B, et al. Gastroenterology. 2010 138:1429-40;Wolchok J D, et al. N Engl J Med. 2017 377:1345-56; Mikucki M, et al.Nat Commun. 2015 6:7458). It is also one of the chemokines in ourearlier reported 12-chemokine gene signature classifier predicting thepresence of the tumor-localized, tertiary lymphoid structures, which arefound to positively correlate with overall survival in certain patientswith metastatic melanoma (Messina J L, et al. Sci Rep. 2012 2:765). Inaddition, CXCL10 has been shown to promote the generation and functionof effector T cells (Dufour J H, et al. J Immunol. 2002 168:3195-204).Given the important role of CXCL10 in mediating T cell recruitment andits positive prognostic value, it seems likely that STING-agonistmediated induction of CXCL10 by tumor cells could further facilitaterecruitment of effector T cells into the tumor microenvironment. Thiscan have at least three important implications. First, it could be usedto recruit higher numbers of T cells into the tumors that lack T cellinfiltration and therefore increase the likelihood of patientsresponding to current immune checkpoint antibody therapies. Second, asame strategy could be used in TIL-based therapies prior to tumorresection and TIL expansion to attract higher numbers of tumor-specificT cells into the tumors with the aim of increasing the probability ofsuccessful expansion of tumor-reactive TIL ex vivo. A third implicationwould also be in adoptive T cell therapy where STING agonist-mediatedCXCL10 induction in tumor cells could be used to improve TIL traffickinginto the tumor sites.

In summary, disclosed herein is the functional significance of the STINGsignaling activation in human melanoma cell lines in response tostimulation with a STING agonist. The disclosed data support that intactactivation of STING signaling in melanomas can promote antitumorimmunity by regulating tumor cell-intrinsic factors that improvetumor-antigen presentation and recognition by immune T cells, as well astheir trafficking.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for enhancing tumor infiltratinglymphocyte (TIL) therapy in a subject, comprising administering to thesubject in need thereof a therapeutically effective amount of acomposition comprising a demethylating agent.
 2. A method for treating amelanoma in a subject, comprising (a) detecting in a biopsy sample fromthe subject reduced STING expression, reduced cGAS expression, or acombination thereof; (b) administering to the subject a therapeuticallyeffective amount of a STING agonist; and (c) administering to thesubject a therapeutically effective amount of a demethylating agent. 3.The method of claim 1, wherein the demethylating agent comprises5-aza-2′-deoxycytidine.
 4. The method of claim 3, wherein the STINGagonist comprises 2′3′-cyclic-GMP-AMP (2′3′-cGAMP).
 5. The method ofclaim 2, further comprising administering to the subject atherapeutically effective amount of a composition comprising HLA-matchedtumor infiltrating lymphocytes (TILs).
 6. A method for producingenhanced tumor infiltrating lymphocytes (TILs), comprising a) culturingtumor cells from a subject in the presence of i) a demethylating agentin an amount effective to demethylate STING proteins in the tumor cellsand ii) a STING agonist in an amount effective to increase theantigenicity of the tumor cells, and b) co-culturing TILs with thesetreated tumor cells and further expanding the TILs after their exposure.7. The method of claim 6, wherein the TILs are HLA matched to a subject.8. A method of treating a subject, comprising producing tumorinfiltrating lymphocytes (TILs) according to the method of claim 6, andadoptively transferring the TILs to the subject.
 9. A method fortreating a melanoma in a subject, comprising (a) detecting in a biopsysample from the subject reduced STING expression, reduced cGASexpression, or a combination thereof; and (b) administering to thesubject a therapeutically effective amount of tumor infiltratinglymphocytes (TILs) produced according to the method of claim
 6. 10. Themethod of claim 8, wherein the subject has a cancer and wherein themethod treats the cancer in the subject.
 11. The method of claim 10,wherein the cancer comprises a melanoma, ovarian cancer, breast cancer,or colorectal cancer.